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Variable Characteristics of Bacteriocin-ProducingStreptococcus salivarius Strains Isolated from MalaysianSubjectsAbdelahhad Barbour, Koshy Philip*
Institute of Biological Sciences, Microbiology Division, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia
Abstract
Background: Salivaricins are bacteriocins produced by Streptococcus salivarius, some strains of which can have significantprobiotic effects. S. salivarius strains were isolated from Malaysian subjects showing variable antimicrobial activity,metabolic profile, antibiotic susceptibility and lantibiotic production.
Methodology/Principal Findings: In this study we report new S. salivarius strains isolated from Malaysian subjects withpotential as probiotics. Safety assessment of these strains included their antibiotic susceptibility and metabolic profiles.Genome sequencing using Illumina’s MiSeq system was performed for both strains NU10 and YU10 and demonstrating theabsence of any known streptococcal virulence determinants indicating that these strains are safe for subsequent use asprobiotics. Strain NU10 was found to harbour genes encoding salivaricins A and 9 while strain YU10 was shown to harbourgenes encoding salivaricins A3, G32, streptin and slnA1 lantibiotic-like protein. Strain GT2 was shown to harbour genesencoding a large non-lantibiotic bacteriocin (salivaricin-MPS). A new medium for maximum biomass production bufferedwith 2-(N-morpholino)ethanesulfonic acid (MES) was developed and showed better biomass accumulation compared withother commercial media. Furthermore, we extracted and purified salivaricin 9 (by strain NU10) and salivaricin G32 (by strainYU10) from S. salivarius cells grown aerobically in this medium. In addition to bacteriocin production, S. salivarius strainsproduced levan-sucrase which was detected by a specific ESI-LC-MS/MS method which indicates additional health benefitsfrom the developed strains.
Conclusion: The current study established the bacteriocin, levan-sucrase production and basic safety features of S. salivariusstrains isolated from healthy Malaysian subjects demonstrating their potential for use as probiotics. A new bacteriocin-production medium was developed with potential scale up application for pharmaceuticals and probiotics from S. salivariusgenerating different lantibiotics. This is relevant for the clinical management of oral cavity and upper respiratory tract in thehuman population.
Citation: Barbour A, Philip K (2014) Variable Characteristics of Bacteriocin-Producing Streptococcus salivarius Strains Isolated from Malaysian Subjects. PLoSONE 9(6): e100541. doi:10.1371/journal.pone.0100541
Editor: Paul D. Cotter, Teagasc Food Research Centre, Ireland
Received January 27, 2014; Accepted May 28, 2014; Published June 18, 2014
Copyright: � 2014 Barbour, Philip. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors wish to acknowledge the support by way of facilities from University of Malaya and the High Impact Research – Malaysian Ministry ofHigher Education grant designated as UM.C/625/1/HIR/MOHE/SC/08 with account F000008-21001 under the Principal Investigator Koshy Philip for the study. Thefunders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* Email: [email protected]
Introduction
Bacteriocin or bacteriocin-like inhibitory substances (BLIS) are
peptide molecules produced by Gram-positive bacteria and some
genera of Gram negative bacteria [1–3]. Lactic acid bacteria are
generally considered to be non-pathogenic (with some exceptions
such as Streptococcus mutans which causes dental caries) and can
produce different kinds of bacteriocins such as nisin produced by
Lactococcus lactis [4–7], plantaricins produced by Lactobacillus
plantarum [8–10], mutacins produced by Streptococcus mutans [11–
15] and salivaricins produced by Streptococcus salivarius [16–20]. S.
salivarius is a species of lactic acid bacteria colonizing the human
oral cavity [21].
Some strains of S. salivarius such as strains K12 and M18 are
now being used as probiotics worldwide due to their capability to
produce different kinds of bacteriocins called lantibiotics
[18,22,23]. Lantibiotics are heat stable ribosomally synthesized
small molecules produced by some strains of gram positive
bacteria with therapeutic potential in treating infectious diseases
[24–29].
To compete better in the oral ecosystem, S. salivarius produce
different kinds of lantibiotics such as salivaricin A, salivaricin B,
salivaricin 9 and salivaricin G32 [16–18,20]. It has been noticed
that bacteriocin or BLIS molecules are not the only useful
metabolites produced by S. salivarius. Levan-sucrase is one of the
important molecules secreted by S. salivarius [30]. Levan-sucrase or
fructosyltransferase (FTF) attack the fructose moiety of sucrose and
polymerize it into fructans which possess levan structure. Levan is
a homo-polysaccharide, non-mutagenic, non-toxic, soluble dietary
fiber with significant prebiotic effects through stimulating the
growth and activity of selected probiotic bacteria in the colon
which can improve the host’s health [31]. Levan may also
PLOS ONE | www.plosone.org 1 June 2014 | Volume 9 | Issue 6 | e100541
Ta
ble
1.
De
ferr
ed
anta
go
nis
mas
say
of
dif
fere
nt
BLI
Sp
rod
uci
ng
S.sa
liva
riu
sst
rain
su
sin
gd
iffe
ren
tp
rod
uct
ion
me
dia
.
Ind
ica
tor
mic
roo
rga
nis
ms
An
tag
on
ism
act
ivit
yo
fS
.sa
liva
riu
sis
ola
tes
tow
ard
sin
dic
ato
rm
icro
org
an
ism
su
sin
gd
iffe
ren
tp
rod
uct
ion
me
dia
NU
10
YU
10
GT
2K
12
12
34
51
23
45
12
34
51
23
45
A.
na
eslu
nd
iiT
G2
22
22
22
22
22
22
22
22
22
22
B.
cere
us
AT
CC
14
57
92
22
22
22
22
22
22
22
22
22
2
Co
ryn
eba
cter
ium
.sp
pG
H1
7++
+++
+++
+2
++++
+++
+++
2++
++2
+++
22
+++
+++
+++
2++
+
E.fa
eciu
mC
1(+
)H(+
)H2
2(+
)H2
22
22
22
22
2++
++++
2++
H.
pa
rain
flu
enza
eT
ON
EJ1
12
22
22
22
22
22
22
22
(+)H
22
22
L.b
ulg
ari
cus
M8
22
22
22
2(+
)H2
22
2(+
)H2
2++
+2
2+
L.la
ctis
AT
CC
11
45
4(+
)H2
22
2(+
)H2
22
22
22
22
+++
+++
++2
++
L.m
on
ocy
tog
enes
NC
TC
+++
++2
++2
2(+
)H2
22
22
22
++
+2
++
M.
lute
us
AT
CC
10
24
0++
+++
+++
+2
+++
++++
+++
+2
+++
++2
+++
2+
+++
+++
+++
2++
+
S.a
ure
us
RF1
22
22
+2
22
22
22
22
22
2++
+++
2(+
)H
S.eq
uis
imili
sA
TC
C1
23
88
++++
+++
+2
+++
+++
+++
2++
+++
+++
+2
2++
+++
+++
+2
+++
S.g
ord
on
iiST
22
22
22
22
22
22
22
22
22
22
2
S.m
uta
ns
GEJ
11
22
22
22
22
22
22
22
2++
++++
2++
S.p
yog
enes
AT
CC
12
34
4++
+++
+++
2++
++
+++
+++
2++
++
+++
+++
2++
+++
+++
+2
+++
S.p
yog
enes
AT
CC
12
34
8++
+++
+++
2++
++
+++
+++
2++
++
+++
++2
2++
+++
+++
+2
+++
S.sa
liva
riu
sG
T2
+++
++2
++
+++
22
22
+2
2++
+++
2++
S.sa
liva
riu
sK
12
22
+2
22
2+
22
22
+2
2+
2+
22
S.sa
liva
riu
sN
U1
02
22
22
22
+2
2+
2+
22
++
+2
+
S.sa
liva
riu
sY
U1
0+
+++
22
(+)H
(+)H
++2
(+)H
22
++2
2++
+++
+2
++
S.sa
ng
uin
isA
TC
C1
05
56
2(+
)H++
+2
(+)H
2(+
)H++
+2
(+)H
2(+
)H++
+2
2++
+++
+++
2(+
)H
W.
con
fusa
A3
22
22
22
22
22
22
22
2+
++
2+
+In
hib
itio
nzo
ne
,0
.75
cm,
++in
hib
itio
nzo
ne
=0
.75
–1
cm,
+++i
nh
ibit
ion
zon
e.
1cm
,2
no
inh
ibit
ion
.H
Haz
yzo
ne
of
inh
ibit
ion
.1T
SYEC
a:T
ryp
tic
Soy
agar
sup
ple
me
nte
dw
ith
2%
Ye
ast
ext
ract
and
0.1
%C
aCO
3.
2B
AC
a:C
olu
mb
iaag
arb
ase
sup
ple
me
nte
dw
ith
5%
wh
ole
de
fib
rin
ate
dsh
ee
pb
loo
dan
d0
.1%
CaC
O3
.3P
TN
YSM
ESm
ed
ium
sup
ple
me
nte
dw
ith
1.5
%b
acte
rio
log
ical
agar
.4C
AB
+NB
CSC
a:C
olu
mb
iaag
arb
ase
sup
ple
me
nte
dw
ith
5%
Ne
wb
orn
calf
seru
man
d0
.1%
CaC
O3
.5M
17
agar
sup
ple
me
nte
dw
ith
2%
Ye
ast
ext
ract
,1
%Su
cro
sean
d0
.1%
CaC
O3
.d
oi:1
0.1
37
1/j
ou
rnal
.po
ne
.01
00
54
1.t
00
1
Lantibiotics Production by Streptococcus salivarius
PLOS ONE | www.plosone.org 2 June 2014 | Volume 9 | Issue 6 | e100541
contribute to human health by its antitumor [32,33] and
antidiabetic activities [34]. Microbial levan is of great importance
for its variable applications in food, cosmetics and pharmaceutical
industries [35]. Levan can also be used in drug delivery
formulations as a coating material and carrier for fragrances and
flavors [36,37].
In this study we describe the different characteristics of strains of
S. salivarius isolated from healthy Malaysian subjects which showed
antagonism against selected Gram-positive bacteria. The prelim-
inary safety assessment study of these strains did not detect any
streptococcal virulence genes and demonstrated the susceptibility
of the S. salivarius strains to a number of classes of antibiotics. The
stability of the metabolic profile was also investigated in this study
and showed some variations among the strains.
A novel method was developed for direct detection of the levan-
sucrase enzyme in crude extracts of S. salivarius cells using LC/MS-
MS technology. The de novo amino acid sequence of the enzyme
showed similarity to that produced by other lantibiotic producing
S. salivarius strains. However, genome sequencing of strain YU10
helped to fully characterise the gene encoding levan-sucrase
production. Due to the high level of levan-sucrase production and
the secretion of lantibiotics, strains presented in this study can play
a great role in pharmaceutical applications as a source of
bacteriocins that can be used as probiotics and/or prebiotics to
improve human oral health. This study also led to the
development of a new medium to obtain higher biomass levels
of S. salivarius and lantibiotic production during aerobic fermen-
tation when compared with other commercial media. This new
medium can be used to enhance bacteriocin production by S.
salivarius which may help to develop new oral probiotics.
Results
1. Antagonism Activity of Bacteriocin Producing S.salivarius
To determine which medium can be used to recover the highest
levels of lantibiotics or bacteriocins, the deferred antagonism assay
was applied using different solid media as a production system.
Strain K12 (salivaricins A and B producer) gave the broadest
antagonism spectrum against a number of selective indicators as
shown in Table 1. BACa and TYECa appeared to be the best
media for lantibiotics production with strain K12. When
PTNYSMES medium was used, strain K12 failed to inhibit the
growth of Lactobacillus delbrueckii subsp. bulgaricus. Strains YU10 and
NU10 inhibited most of the streptococcal strains used in this study
(but not Streptococcus mutans) while the levels of lantibiotics secreted
by these strains were improved when PTNYSMES medium was
used as the production medium. The inhibitory spectrum of both
NU10 and YU10 included one Listeria monocytogenes strain (partial
inhibition). When blood was used as a supplementary component
in the production medium (BACa), strain GT2 expressed S.
pyogenes-inhibitory activity. Surprisingly, no anti-Micrococcus luteus
inhibitory activity was detected when strain GT2 was grown onto
BACa plates indicating that the bacteriocin produced may not be
a lantibiotic since M. luteus is known for its extreme susceptibility to
lantibiotics. However, when GT2 was grown on PTNYSMES and
TSYECa media, there was some inhibition towards M. luteus. In all
media used with strain GT2 as a producer, most of the inhibitory
activity was eliminated when the media were heated at 70uC for
30 minutes (data not shown). This finding indicates that strain
GT2 may express heat labile bacteriocin. It was noticed that strain
YU10 does not exhibit self-immunity as it showed significant
antagonism activity towards itself when tested for self-immunity
assay with all kinds of media. However, when PTNYSMES was
used in this test, most of the producers showed lack of self-
immunity (Table 1).
2. Genes Encoding Salivaricins ProductionStrain K12 is known to harbour the structural genes salA, sboA
and MPS var encoding salivaricins A, B and MPS variant
production respectively while no sivA, MPS or slnA were detected
within this strain. Strains NU10 and YU10 were shown previously
to harbour sivA (structural gene for salivaricin 9 production) and
salA [38]. In this study, strain YU10 was found to harbour
structural genes encoding for streptin, salivaricin A3 and
salivaricin G32 lantibiotics and the slnA1 lantibiotic-like protein.
However, salivaricin G32 was the only bioactive lantibiotic
expressed and detected in strain YU10 and it seems that not all
of these genes are likely to be expressed. Genome sequencing of
strain YU10 confirmed the presence of genes mentioned above. It
was found that salivaricin A is quite widely distributed in
streptococcal species but the locus is often quite defective and so
the inhibitory product is not expressed even though the salivaricin
A immunity component may be functional. Strain GT2 was shown
to harbour MPS and MPS var genes encoding the production of
salivaricin MPS and MPS variant respectively.
3. Metabolic Profiles and Biochemical Characteristics of S.salivarius Isolates
The stability of the metabolic profiles of S. salivarius strains was
carried out using API kits. The biochemical characteristics of each
strain provided important information on the needs and criteria of
each isolate to achieve optimal growth. YU10 was the only strain
tested in this study that showed D-sorbitol positive reaction. This
information can also be used as a differentiation method for YU10
detection. Unlike NU10 and YU10, strains K12 and GT2 are able
to use inulin or galactose as a carbon source. NU10 was the only
strain with positive reaction for amygdalin while GT2 was the only
strain that fermented melibiose. Strain K12 also differed with the
other strains by fermenting D-tagatose. NU10 was the only strain
that failed to ferment lactose and this is unusual for lactic acid
bacteria. However, when lactose was used as a carbon source
during media development, strain NU10 was unable to grow
adequately. YU10 was the only strain with trehalose negative
reaction (Table 2).
4. Antibiotics SusceptibilityS. salivarius isolates tested in this study were assessed to be
moderately resistant to gentamicin and this finding is similar to
what was published for strain K12 and other S. salivarius isolates
[39]. According to CLSI breakpoints for ofloxacin, all S. salivarius
strains tested in this study were sensitive to this antibiotic
(inhibition zone .16 mm). Furthermore, S. salivarius strains tested
in this study were sensitive to several routinely used antibiotics for
the control of upper respiratory tract infections. It was noticed that
strain YU10 showed intermediate susceptibility levels to erythro-
mycin with 19.7 mm zone of inhibition. However, the other
strains NU10, GT2 and K12 were susceptible to the same
antibiotic. No significant differences regarding antibiotic suscep-
tibility were observed after two years of storage indicating that the
strains tested in this study are reasonably stable. Full results of
antibiogram are listed in Table 3.
5. Genome Sequencing of Strains NU10 and YU10Genome sequencing of strains NU10 and YU10 was performed
with Illumina Miseq sequencer. Total of 2,344,494 and 2,345,259
pair-end reads were generated with contigs numbering 51 and 48
Lantibiotics Production by Streptococcus salivarius
PLOS ONE | www.plosone.org 3 June 2014 | Volume 9 | Issue 6 | e100541
Ta
ble
2.
AP
I2
0ST
REP
and
AP
I5
0C
HL
reac
tio
ns
for
S.sa
liva
riu
sis
ola
tes.
AP
I2
0S
TR
EP
K1
2N
U1
0N
U1
0V
YU
10
YU
10
VG
T2
GT
2V
Ace
toin
pro
du
ctio
n+
++
++
++
Hyd
roly
sis
(HIP
pu
ric
acid
)2
22
22
22
B-G
luco
sid
ase
hyd
roly
sis
(Esc
ulin
)+
++
++
++
Pyr
rolid
on
ylar
ylam
idas
e2
22
22
22
a-G
alac
tosi
das
e2
22
22
2+
b-G
lucu
ron
idas
e2
22
22
22
b-G
alac
tosi
das
e2
22
22
22
Alk
alin
ep
ho
sph
atas
e+
+2
++
++
Leu
cin
eam
ino
pe
pti
das
e+
++
++
++
Arg
inin
ed
ihyd
rola
se2
22
22
22
D-R
ibo
se2
22
22
22
L-A
rab
ino
se2
22
22
22
D-M
ann
ito
l2
22
22
22
D-S
orb
ito
l2
22
++
22
D-L
acto
se+
22
++
22
D-T
reh
alo
se+
++
22
++
Inu
lin+
22
22
-+
D-R
affi
no
se+
++
++
++
star
ch+
++
++
++
gly
cog
en
22
22
22
2
AP
I5
0C
HL
K1
2N
U1
0N
U1
0V
YU
10
YU
10
VG
T2
GT
2V
L-A
rab
ino
se2
22
22
++
Rib
ose
22
22
2+
+
Gal
acto
se+
22
22
++
D-G
luco
se+
++
++
++
D-F
ruct
ose
++
++
++
+
D-M
ann
ose
++
++
++
+
Sorb
ito
l2
22
++
22
a-M
eth
yl-D
-glu
cosi
de
22
22
22
2
N-A
cety
lg
luco
sam
ine
++
++
+2
2
Am
ygd
alin
2+
+2
22
2
Arb
uti
n+
++
++
++
Escu
lin+
++
++
++
Salic
in+
++
++
++
Lantibiotics Production by Streptococcus salivarius
PLOS ONE | www.plosone.org 4 June 2014 | Volume 9 | Issue 6 | e100541
for NU10 and YU10 respectively. Genome annotation was
performed using RAST [40]. A total of 2146 coding sequences
(CDSs) and 38 structural RNAs were predicted in strain NU10
while 2161 CDSs and 41 structural RNAs were predicted in strain
YU10. Genes encoding for streptin, salivaricin A3, salivaricin G32
lantibiotics and slnA 1 lantibiotic-like protein were detected in the
genome draft of strain YU10. However, only salivaricin A3,
salivaricin G32 and slnA 1 lantibiotics showed 100% homology to
genes present in strain YU10. The gene encoding for streptin
present in YU10 genome has only 82% homology to streptin due
to some mutations (Text S1). Furthermore, strains YU10 and
NU10 were shown to be free of any streptococcal pyrogenic
exotoxins, streptococcal superantigen A (SSA), streptococcal
mitogenic exotoxin Z (SmeZ) and streptodomase B. Other
streptococcal virulence factors listed in Table 4 were also
investigated and none of them were present in YU10 or NU10
genomes. Figure 1 shows subsystem feature counts of S. salivarius
strains NU10 and YU10 detected by RAST. Under virulence,
disease and defense category we can clearly observe that no
Streptococcus pyogenes virulence determinants, toxins or superantigens
were detected. Other factors related to bacteriocin production,
protein synthesis and adhesion factors are not part of the toxin and
virulence factors. This assessment suggests that both YU10 and
NU10 are safe for future use as probiotics.
6. Developing New Bacteriocin-production MediumThe newly developed medium used in this study helped to
increase the biomass of S. salivarius cultures grown aerobically. The
carbon source (sucrose) used within this medium was adequate to
grow all the strains as some of them cannot process lactose (strain
NU10). Usually, S. salivarius requires CO2 enriched atmosphere to
grow adequately and thereby produce lantibiotic molecules.
However, in this study we tried to develop enriched medium that
helped to cultivate S. salivarius aerobically without any supple-
mented CO2. However, strain NU10 showed some susceptibility
to levels of CO2 (3–5%) and it did not grow in M17 medium
(Merck) which is supplemented originally with lactose as a carbon
source. This finding confirms the metabolic profile of strain NU10
which was unable to uptake lactose. Strain GT2 also showed weak
growth in M17 medium used in this study and this event cannot be
linked to carbon source since this strain showed positive reaction
for lactose test. When THB or BHI media were used, some lytic
activities were observed after 20 hours of bacterial growth as the
OD600 values started to decrease. Although it is designed for lactic
acid bacteria, MRS medium failed to grow S. salivarius. However,
strain GT2 showed better growth (but still weak, OD600 = 0.4)
when grown in this medium as compared with other S. salivarius
strains. YNS medium showed better bacterial growth compared to
other commercial media especially for strains GT2 and K12.
However, the newly developed PTNYSMES was the best medium
tested for S. salivarius growth in this study and showed a significant
increase in the optical density of all the isolates. Compositions of
all media used are listed in Table 5. The differences in pH values
before and after 22 hours of fermentation for each medium are
listed in Table 6. All isolates reached the stationary phase of
growth in just 10 hours and showed no autolytic activities even
after 24 hours. OD600 = 1 was achieved with strains K12, NU10
and GT2 while strain YU10 also showed good biomass
accumulation with OD600 = 0.9 (Figure 2).
7. Salivaricin 9 and Salivaricin G32 ProductionAttempts to recover lantibiotics from S. salivarius cells grown in
PTNYSMES medium were successful. Both strains NU10 and
YU10 were grown for 24 hours in this medium and the lantibiotics
Ta
ble
2.
Co
nt.
AP
I2
0S
TR
EP
K1
2N
U1
0N
U1
0V
YU
10
YU
10
VG
T2
GT
2V
Ce
llob
iose
++
++
++
+
Mal
tose
++
++
++
+
Lact
ose
+2
2+
++
+
Me
libio
se2
22
22
++
Sacc
har
ose
++
++
++
+
Tre
hal
ose
++
+2
2+
+
Inu
lin+
++
++
++
Me
lezi
tose
22
22
22
2
D-R
affi
no
se+
++
++
++
b-G
en
tio
bio
se2
++
++
++
D-T
agat
ose
+2
22
22
2
VSt
rain
was
sub
-cu
ltu
red
sub
seq
ue
ntl
yfo
r2
0ti
me
sb
efo
rete
stin
gfo
rm
eta
bo
licp
rofi
le.
do
i:10
.13
71
/jo
urn
al.p
on
e.0
10
05
41
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Lantibiotics Production by Streptococcus salivarius
PLOS ONE | www.plosone.org 5 June 2014 | Volume 9 | Issue 6 | e100541
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Lantibiotics Production by Streptococcus salivarius
PLOS ONE | www.plosone.org 6 June 2014 | Volume 9 | Issue 6 | e100541
were subsequently recovered by cell extraction followed by further
chromatography techniques for lantibiotic purification. MALDI-
TOF (MS) analysis showed that like our previous report [38],
salivaricin 9 (2560 Da) was produced by strain NU10 using
PTNYSMES medium in the current study. Furthermore, salivar-
icin G32 (2667 Da) (Figure 3) was the only detectable and known
lantibiotic produced by this strain when grown in the new
medium. Salivaricin A was not produced or detected by strains
YU10 or NU10 using this medium even though the strains
harbour the structural gene encoding this lantibiotic. The
production experiment was repeated without adjusting the pH of
the medium after fermentation (without adsorption) to calculate
levels of lantibiotics attached to the producer cells. However,
attempts to recover lantibiotics from the cell-free supernatant of
this preparation using 80% ammonium sulphate saturation as
described previously [41] showed that 60–70% of lantibiotic
produced by NU10, YU10 and K12 strains presented in this study
is cell-wall associated peptide (Table 7). The bacteriocin units
(arbitrary units) were calculated as mentioned previously [38].
8. Levan-sucrase Detection and CharacterizationCell-associated levan-sucrase was extracted from S. salivarius
cells of strain YU10. Advanced LC-MS/MS method was
developed for direct detection of this unique enzyme from the
cell extract using reverse phase chromatography. The peptide
which matched the levan-sucrase enzyme (accession: Q55242)
contains 14 residues (VGTLAFLGATQVKA). The match was
considered significant by the search algorithm with a score of
78.88 and coverage of 1.44. This defines matches with ion score of
51 for identity and charge of 2. Retention time for levan-sucrase
was 38.71 minutes with MH+ [Da] = 1375.79582 (Figure 4).
Genome sequencing of strain YU10 revealed the structural gene
encoding for levan-sucrase or fructosyltransferase (FTF) produc-
tion. Full characterization of the gene (ftf) with in silico protein
translation is provided as support information (Text S2). The ftf
region of strain YU10 was compared to ftf region in the
commercial probiotic strain M18 genome [23] and both regions
were almost identical. In addition to fructosyltransferase, this
region included gene encoding for levanase production (Figure
S1).
Discussion
Three different salivaricin-producing S. salivarius strains isolated
from Malaysian subjects were evaluated in this study and shown to
produce different kinds of BLIS molecules some of which are
lantibiotics (sal9 and salG32). Gene encoding a large peptide
molecule salMPS (accession number: AGBV01000006) was also
detected in one of the strains (GT2).
Strains K12, NU10 and YU10 produced inhibitory activity
when grown on different media including M17 (Difco), BACa,
PTNYMES and others mentioned in the results. On the other
Figure 1. Subsystem feature counts of S. salivarius strains NU10 and YU10 detected by RAST. No S. pyogenes virulence determinants weredetected.doi:10.1371/journal.pone.0100541.g001
Lantibiotics Production by Streptococcus salivarius
PLOS ONE | www.plosone.org 7 June 2014 | Volume 9 | Issue 6 | e100541
hand, strain GT2 failed to produce significant anti-S. pyogenes
inhibitory activity when grown on media which was not
supplemented with blood but produced significant inhibitory
activity against S. pyogenes when grown on BACa. This indicates
that the production of anti-S. pyogenes inhibitory activity by this
strain is likely to be dependent on blood components. This
characteristic is similar to salivaricin MPS-like peptide which is a
large bacteriocin molecule [42]. Further analysis showed that
strain GT2 harbours the structural gene encoding for salivaricins
MPS and MPS variant productions.
Strain NU10 was shown to harbour structural genes encoding
salivaricins A and 9 previously but only sal9 could be produced
and detected as an active peptide in the present study. Strain
YU10 was shown to harbour genes encoding salivaricins A3, G32,
streptin and slnA1 lantibiotic-like protein, however, only salG32
was detected and recovered from this strain.
The strains in this study also showed some variations in their
metabolic profiles. Surprisingly, strain NU10 showed a negative
reaction for lactose fermentation and when the strain was
propagated in growth medium containing lactose as the only
carbon source, it showed significantly weaker growth and total
absence of any lantibiotic production. A previous study that was
done in our laboratory [38] showed that this strain is a producer of
salivaricin 9. The maximum yield of BLIS activities was recovered
when sucrose was used as the carbon source.
The use of commercial media including THB and BHI in
aerobic condition resulted in a drop of OD600 reading that is
apparently attributed to microbial cell lysis. The reason for this
Table 4. Virulence assessment for S. salivarius strains YU10 and NU10.
Virulence determinant Gene designation S. salivarius strains
YU10 NU10
M-protein emm – –
Protein H sph – –
Streptokinase Ska – –
CAMP factor cfa – –
Streptolysin S SagA – –
Streptolysin O slo – –
Hyaluronate lyase hyl – –
Nicotin adenine dinuclutide glycohydrolase nga – –
Streptococcal pyrogenic exotoxin A SpeA – –
Streptococcal pyrogenic exotoxin B SpeB – –
Streptococcal pyrogenic exotoxin C SpeC – –
Streptococcal pyrogenic exotoxin G SpeG – –
Streptococcal pyrogenic exotoxin H SpeH – –
Streptococcal pyrogenic exotoxin I SpeI – –
Streptococcal pyrogenic exotoxin J SpeJ – –
Streptococcal pyrogenic exotoxin K SpeK – –
Streptococcal pyrogenic exotoxin L SpeL – –
Streptococcal pyrogenic exotoxin M SpeM – –
Streptococcal superantigen A SSA – –
Streptococcal metogenic exotoxin Z SmeZ – –
Streptodornase B SdaB – –
Fibrinogen binding protein fba – –
Fibrotectin-binding protein (protein F) prtF – –
Protein G-related alpha 2 macroglobulin binding protein grab – –
Streptococcal inhibitor of complement SIC – –
Immunoglobulin G-endopeptidase IdeS – –
Secreted endo-b-N-acetylglucosaminidase ndoS – –
C5a peptidase ScpA – –
Fibronectin-binding protein FBP – –
Serum opacity factor SOF – –
C3 family ADP-ribosyltransferase SpyA – –
Serine endopeptidase ScpC – –
Hyaluronan synthase HasA – –
Collagen-like surface protein SclB – –
(2): absence of the virulence factor.doi:10.1371/journal.pone.0100541.t004
Lantibiotics Production by Streptococcus salivarius
PLOS ONE | www.plosone.org 8 June 2014 | Volume 9 | Issue 6 | e100541
Figure 2. Growth kinetics of S. salivarius strains grown aerobically in different media. A: THB (BD), B: BHI (BD), C: M17 (Merck), D: MRS(Merck), E: YNS and F: PTNYSMES.doi:10.1371/journal.pone.0100541.g002
Lantibiotics Production by Streptococcus salivarius
PLOS ONE | www.plosone.org 9 June 2014 | Volume 9 | Issue 6 | e100541
Ta
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Lantibiotics Production by Streptococcus salivarius
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lysis in aerobic condition is still unknown and perhaps the aerobic
condition is not ideal for strain K12 and other S. salivarius isolates
when THB or BHI media are used for propagation (Figure 2). In
the current study, a newly developed medium buffered with MES
helped to enhance the biomass and bacteriocin production by S.
salivarius which grew well in an aerobic atmosphere. This finding
Table 6. Variation of the pH values of S. salivarius cultures grown in different media after 22 hours of growth.
Medium Initial pH of the medium/final pH of the culture after 22 h fermentation
K12 GT2 NU10 YU10
THB 7.8/5.00 7.8/5.08 7.8/5.16 7.8/5.23
BHI 7.01/5.16 7.01/5.38 7.01/5.33 7.01/5.64
M17 7.13/5.57 7.13/6.55 7.13/6.82 7.13/5.74
MRS 5.59/4.90 5.59/5.00 5.59/5.03 5.59/5.59
Brucella Broth 6.53/5.09 6.53/5.14 6.53/5.08 6.53/5.11
YNS 6.71/3.83 6.71/3.78 6.71/3.84 6.71/3.91
PTNYSMES 6.51/4.31 6.51/4.30 6.51/4.27 6.51/4.33
doi:10.1371/journal.pone.0100541.t006
Figure 3. Purification and detection of salivaricin G32 produced by strain YU10 grown in PTNYSMES. A: Cation exchangechromatography of the cell extract using SP FF column, B1: RP HPLC of the pooled active fractions of salivaricin G32 obtained from A, B2: second RPHPLC of the active fraction obtained by B1. C: MALDI-TOF (MS) analysis of the pure salivaricin G32.doi:10.1371/journal.pone.0100541.g003
Lantibiotics Production by Streptococcus salivarius
PLOS ONE | www.plosone.org 11 June 2014 | Volume 9 | Issue 6 | e100541
can solve the problem of scaling-up the culture in large scale
bioreactors for probiotic and/or lantibiotic production. Previous
study showed that buffering the medium with MES helped to
achieve higher biomass levels of Streptococcus thermophilus [43]. Using
organic buffers for bacteriocin production helps to prevent
extreme drop in pH of medium due to the production of lactic
acid or other substances.
It has been noticed that 60–70% of the bacteriocins recovered
in this study were cell-wall associated peptides bound to the
producer cells while the rest of the inhibitory peptides were
secreted extracellularly into the liquid media. Cell-associated
bacteriocins produced by lactic acid bacteria had been reported
previously [44,45]. Hence, this class of bacteriocins can be
recovered from producer cells grown in liquid media.
Most lantibiotics appear to be regulated at the transcriptional
level in a cell-density-dependent manner in various bacteria [46].
The mode of regulation for lantibiotic production has been shown
to involve secreted peptides that act as communication molecules
accumulated in the environment during growth. When certain
concentrations of these molecules are reached, high level of
lantibiotic production is triggered [46]. A previous study
demonstrated that the lantibiotic produced by strain NU10 is
auto-regulated and the same lantibiotic could induce its produc-
tion by strain NU10 [38]. However, strain NU10 was also shown
to encode structural genes for salivaricins A and 9. But it was
obvious that when an enhanced culture of strain NU10 was
analysed using MALDI-TOF MS, salivaricin 9 was the only
lantibiotic detected from the purified supernatant. Hence, we can
conclude that the presence of structural genes encoding produc-
tion of salivaricins in S. salivarius strains does not necessarily mean
that the bioactive molecule is expressed or that the PTNYSMES
medium used for the production in aerobic condition did not
support the biosynthesis of that particular peptide.
Strain YU10 was shown to produce salG32 while no salivaricin
A, 9 or streptin production was detected. Previous work showed
that in contrast to the regulation of sal9, the signal of up-regulation
Table 7. Inhibitory activity recovered from cell extracts and cell-free supernatants of S. salivarius cultures.
Inhibitory activity recovery (From 1 Lculture)1 Lantibiotic-producing S. salivarius strains
NU10 YU10 K12
Total activity 4.86104 AU (100%) 1.326104 AU (100%) 1.026105 AU (100%)
Cell extract activity 3.26104 AU (66.6%) 9.66103 AU (72.7%) 6.46104 AU (62.5%)
Cell-free supernatant activity 1.66104 AU (33.4%) 3.66103 AU (27.2%) 3.846104 AU (37.5%)
1Strains were grown in PTNYSMES medium.AU: arbitrary unit.doi:10.1371/journal.pone.0100541.t007
Figure 4. Detection of levan-sucrase enzyme produced by strain YU10 using ESI-LC-MS/MS analysis.doi:10.1371/journal.pone.0100541.g004
Lantibiotics Production by Streptococcus salivarius
PLOS ONE | www.plosone.org 12 June 2014 | Volume 9 | Issue 6 | e100541
of salivaricin G32 is not the antimicrobial peptide itself but rather
some other substances produced by the lantibiotic producer [20].
The variety of bacteriocins produced by S. salivarius isolated
from Malaysian subjects makes it interesting to study these
molecules and their distribution among Malaysian population.
High throughput genome sequencing of both strains NU10 and
YU10 using Illumina’s MiSeq genome sequencing confirmed the
absence of the streptococcal virulence determinants within both
genomes (Figure 1) (Table 4). This finding nominates some of these
strains as potentials for probiotic development as they pass the
initial safety assessments described previously for S. salivarius strain
K12 [39].
Bacteriocins and lantibiotics were not the only unique and
useful molecules being produced by the strains described in this
study. When sucrose was added to the medium as the only source
of carbon, levan-sucrase enzyme was produced in significant levels.
Levan-sucrase (fructosyltransferase) is a very unique cell-bound
enzyme produced by S. salivarius and it plays an important role in
the production of levan residues. Levan has been shown to have
prebiotic effects and so this production, together with the
production of lantibiotics, makes the strain potentially useful for
multiple applications. The method described in this study for
direct detection of levan-sucrase from the cell-extract using LC-
MS/MS was efficient to detect levan-sucrase in S. salivarius and the
full characterization of the gene encoding levan-sucrase produc-
tion was elucidated using genome sequencing of the producer
strain.
In conclusion, S. salivarius strains evaluated in this study showed
critical variations in the type of inhibitory substances produced
some of which are lantibiotics sal9 and salG32 produced by strains
NU10 and YU10 respectively while gene encoding large
bacteriocin molecule salMPS was detected in strain GT2. No
significant variations in antibiotic susceptibility among S. salivarius
isolates were observed after two years of storage indicating stability
of the strains in terms of susceptibility towards antibiotics. The
metabolic profile studies showed some variations among the tested
strains and gave important information on the biochemical criteria
required by each strain to perform better during fermentation
studies. The in vitro safety assessment tests showed that the strains
are free of virulence genes known to be present in streptococcal
pathogens and this finding was supported by genome sequencing
of strains NU10 and YU10.
Strains NU10 and YU10 produce sal9 and salG32 lantibiotics
respectively which distinguish from the well characterised S.
salivarius probiotic strain K12 producing the lantibiotics salA and
salB. These differences introduce additional options for probiotics
that may be used in oral health management with different
lantibiotic molecules.
The developed medium PTNYSMES helped to enhance
biomass accumulation of all strains and attempts to recover
lantibiotics produced by S. salivarius grown in this medium
aerobically were successful. A new method for levan-sucrase
detection was also developed and gene encoding levan-sucrase
production was characterized. The ability of S. salivarius to produce
lantibiotics and levan-sucrase adds value to this microorganism
with dual benefits for probiotic development with prebiotic effects.
Materials and Methods
1. Bacterial Strains and Culture MediaS. salivarius strains NU10, YU10 and GT2 were isolated from
the oral cavity of healthy Malaysian subjects and were deposited in
the NCBI gene bank under accession numbers KC796011,
KC796012 and KC796010 respectively. S. salivarius strain K12
was kindly provided by John Tagg (University of Otago, BLIS
Technologies, New Zealand). Indicator strains including Bacillus
cereus ATCC14579, Lactococcus lactis ATCC11454, Micrococcus luteus
ATCC10240, Streptococcus dysgalactiae subsp. equisimilis
ATCC12388, Streptococcus pyogenes ATCC12344, Streptococcus pyo-
genes ATCC12348, Streptococcus sanguinis ATCC10556 were pur-
chased from American Type Culture Collection (ATCC). Listeria
monocytogenes NCTC10890 was purchased from National Collec-
tion of Type Cultures. Other indicator strains such as Actinomyces
naeslundii TG2, Corynebacterium spp GH17, Enterococcus faecium C1,
Haemophilus parainfluenza TONEJ11, Lactobacillus bulgaricus M8,
Staphylococcus aureus RF122, Streptococcus gordonii ST2, Streptococcus
mutans GEJ11, Weissella confusa A3 were taken from the culture
collection of Microbial Biotechnology Laboratory, Division of
Microbiology, Institute of Biological Science, Faculty of Science,
University of Malaya, Kuala Lumpur, Malaysia. Todd Hewitt
broth (THB) (Difco) was used to propagate all the bacterial strains
in this study. Mitis Salivarius agar (MSA) (Difco) was used to
isolate pure colonies of S. salivarius strains. M17 (Merck), MRS
(Merck), Brain Heart Infusion (BHI) (Difco) media were used to
study the growth kinetics of S. salivarius strains. Columbia blood
agar base (Difco) supplemented with 5% defibrinated sheep blood
and 0.1% CaCO3 (BACa) was used to carry out the deferred
antagonism test.
2. Ethical Approval for S. salivarius SamplingThe subjects were required to sign a consent form to isolate S.
salivarius from the tongue surface using sterile cotton swab.
Approval for sampling from tongue surface is not required from
the IRB as a proforma for written consent from the subject is
approved by the IRB. The ethics committee IRB Reference
Number is DF OP1304/0019 (P) for our Institution (University of
Malaya). This was discussed with the ethics committee (IRB) and
the protocol used complied with Good Laboratory Practices.
Therefore IRB approval is not required prior to sampling in this
investigation.
3. Deferred Antagonism TestThe method was first described by [47] and performed in this
study with some modifications. Bacteriocin producer strains were
streaked across different production media agar plates as 1 cm
wide strip using sterilized cotton swabs. The producers were then
incubated aerobically with 5% CO2 for 18 hours at 37uC. Sterile
cotton swabs were used to remove the bacteriocin producer
bacteria before the plates were sterilized by inverting the plates
over filter paper soaked with chloroform for 30 minutes. The
plates were aired for another 30 minutes to get rid of any
chloroform residues. Indicator bacterial strains of OD600 = 0.1
were streaked at a right angle across the producer streak. The
plates were re-incubated under the same conditions mentioned
above for 18 hours. Zones of no bacterial growth were recorded as
antagonism activity due to bacteriocin production (Table 1). The
antagonism assay for each strain was repeated twice using different
production media.
4. DNA Extraction and Distribution of Genes EncodingSalivaricins Production
Single S. salivarius colonies grown on MSA medium for 18 hours
were transferred to 10 ml of sterilized THB and incubated
aerobically with 5% CO2 for 18 hours at 37uC before the pellets
were centrifuged at 80006g for 5 min at 4uC and suspended in
400 ml of 0.85% NaCl in water (w/v). The suspension was heated
at 70uC for 30 min and the bacterial pellets were collected at
Lantibiotics Production by Streptococcus salivarius
PLOS ONE | www.plosone.org 13 June 2014 | Volume 9 | Issue 6 | e100541
80006g for 5 min at 4uC. The pellets were suspended in lysing
buffer (2 mM Tris-HCl pH 8, 0.2 mM EDTA, 1.5% Triton X100
and 1000 U of mutanolysin) and incubated at 37uC for 2 hours.
50 mg/ml of lysozyme was added and the samples were further
incubated for one hour at 37uC. After three freeze-thaw cycles, the
samples were then processed using DNeasy Blood and Tissue kit
(Qiagen) following manufacturer’s instructions for DNA extraction
from Gram-Positive bacteria. PCR conditions for salivaricin genes
amplifications were applied as described previously [16,17,20,48]
with some modifications to the reaction composition which
included using Top Taq Master Mix (QIAGEN). This experiment
was performed using Applied Biosystems Veriti 96-Well Thermo
Cycler.
5. Biochemical Characterization of S. salivarius IsolatesAll the biochemical tests were performed using 50 CH and 20
strep API kits to study the metabolic profiles of S. salivarius strains
as shown in Table 2. The API kits were used according to
manufacturer’s instructions (API-bioMeriex).
6. Antibiotics Susceptibility TestThe antibiograms of the three strains NU10, YU10 and GT2
were tested by the antibiotic disc diffusion assay according to
Clinical and Laboratory Standards Institute (CLSI) (Vol. 32, No.3,
Jan, 2012). S. salivarius cultures were grown on Mueller Hinton
Agar (Difco, USA) supplemented with 5% sheep blood (Liofilchem
srl, Italy) for 20 hours at 37uC in 5% CO2 atmosphere using BD
Gas Pak EZ CO2 container system. Bacterial suspensions were
prepared from morphologically identical colonies grown on the
agar plates and suspended in saline solution (0.85% NaCl in
water). The resultant bacterial suspensions were adjusted to
turbidity of 0.5 McFarland before bacterial lawns were performed
on the same blood agar plates mentioned above. Antimicrobial
susceptibility test discs (OXOID, UK) were placed on top of the
pre seeded plates using sterile forceps and the plates were
incubated as mentioned above. Antibiotics used in this test are
penicillin G, penicillin V, amoxicillin, ofloxacin, tetracycline,
erythromycin, gentamicin, clindamycin, streptomycin, vancomy-
cin, novobiocin and chloramphenicol. Both original cultures
stored at 280uC from two years and weekly subcultures used in
the lab routinely were tested to check for any differences following
storage. Strain K12 was also tested as a control since it was
reported previously [39] for susceptibility against the same and
additional antibiotics used in this study. Measurement of the
diameters of zones of complete inhibition (as judged by the
unaided eye) including the diameter of the disc are listed in
Table 3. This experiment was repeated in triplicates and showed
almost identical results.
7. Preparation of the First Genome Drafts for StrainsNU10 and YU10
Genome sequencing was carried out using Illumina’s compact
MiSeq system at the High Impact Research Center, University of
Malaya, Malaysia. Genomic libraries were prepared using the
Nextera kit Illumina (Illumina, Inc., San Diego, CA) which
produced a mean insert size between 800 and 1,200 bp. Total of
379-fold and 204-fold coverages were generated for strains NU10
and YU10 respectively. Approximately 85% of these reads were
assembled using CLC Bio Genomic Workbench Software Version
6.0.5. Genome annotation was performed using RAST Version
4.0 [40]. The genome analysis included the virulence assessment
for YU10 and NU10 strains to prove the absence of any
streptococcal virulence determinants within both strains genomes
(Table 4).
8. Developing New Bacteriocin-production MediumDifferent media e.g. M17, MRS, THB and BHI were used to
study the growth kinetics of S. salivarius grown aerobically at 37uC.
Each S. salivarius strain was grown on BACa plates (Columbia agar
base supplemented with 5% whole human blood and 0.1%
CaCO3 for 18 hours at 37uC. Then the bacteria was washed from
the agar plates using phosphate buffer saline at pH 7 and
centrifuged to pellet the cells at 50006g for 10 minutes. Bacterial
pellets were washed twice with the same buffer using the same
conditions mentioned above before re-suspending in the same
buffer. The bacterial suspension was then diluted using the same
buffer to 0.5 McFarland before 20 ml of each bacterial suspension
was used to inoculate 180 ml of each medium into a 96-well sterile
plate. The plate used in this experiment was flat base well plate
and covered with a sterile plastic lid to prevent contamination.
The growth was monitored by measuring the OD at a wavelength
of 600 nm using a Multiskan GO Microplate Spectrophotometer
(Thermo Scientific) over 24 hours. The spectrophotometer was set
at medium speed shaking for 20 seconds before each photometric
measurement. This experiment was carried out in triplicates and
average of each triplicate measurement was used for growth
kinetics (Figure 2). YNS medium (1% yeast extract, 1%
neopeptone and 1% sucrose) and PTNYMES medium (1%
peptone, 1% tryptone, 1% neopeptone, 1% yeast extract, 1%
sucrose, 1% MES (2-(N-morpholino)ethanesulfonic acid), 0.2 g/L
NaCl, 0.5 g/L ascorbic acid, 0.25 g/L magnesium sulphate and
0.2 g/L sodium acetate) were also used in this study. Typical
compositions of all media used in this study are listed in Table 5.
9. Lantibiotics Production and PurificationPTNYMES medium (adjusted before autoclaving at pH 6.5
using concentrated NaOH) was inoculated with 5% of S. salivarius
cultures grown for 18 hours in the same medium. 4000 mL
shaking flasks were used for this experiment at 37uC for 22 hours
with 150 rpm orbital shaking aerobically. The cultures were
adjusted to pH 5.8 and incubated for 1 hour at 4uC to adsorb
levels of lantibiotics secreted into the liquid medium to the
producer’s cells. Then the cultures were centrifuged at 85006g for
30 minutes and the cells were re-suspended in 95% methanol
(adjusted to pH 2 by concentrated HCl). The cell suspensions were
stirred gently overnight at 4uC for 18 hours before the cells were
collected by centrifugation at 10006g for 30 minutes. The
supernatant was evaporated using a rotary evaporator at 45uCand the crude lantibiotic was assayed for antimicrobial activity.
The crude preparation was concentrated 10 fold and then diluted
1:5 (v/v) with 20 mM sodium phosphate buffer pH 5.8. This final
preparation was subjected to FPLC AKTA purifier (GE
Healthcare) using HiTrap SP FF strong cation exchanger column
pre-equilibrated with 20 mM sodium phosphate buffer pH 5.8
(buffer A). The column was washed with 10X column volume of
buffer A before a leaner gradient of buffer B (1 M NaCl in buffer
A) was applied. Eluted fractions were collected using auto collector
and the separation was monitored using three different UV wave
lengths (207, 214 and 280 nm). Active fractions from 5 FPLC runs
were pooled and concentrated before subjecting to Chromolith
SemiPrep RP-18e 100-10 mm column using Waters HPLC system
with gradient of 20% to 50% acetonitrile in water (v/v). UV
wavelength of 214 nm was used to detect peptide peaks and the
active fraction was subjected for the second time using the same
column and conditions mentioned above to obtain the pure
peptide. Well diffusion assay was performed to identify biologically
Lantibiotics Production by Streptococcus salivarius
PLOS ONE | www.plosone.org 14 June 2014 | Volume 9 | Issue 6 | e100541
active fractions using Micrococcus luteus ATCC10240 and Streptococ-
cus pyogenes ATCC12344 as indicator targets. Pure lantibiotics were
subjected to matrix assisted laser desorption ionization time of
flight mass spectrometry MALDI-TOF (MS) using 4800 Plus
MALDI TOF/TOF Analyzer to determine the molecular weight
of the lantibiotic (Figure 3).
10. Production of LevansucraseStrain YU10 was grown aerobically with 5% CO2 for 18 hours
at 37uC in one litre of M17 medium supplemented with 2% yeast
extract, 2% sucrose and 0.1% CaCO3 (M17YESUCa). The cells
were collected by centrifugation at 180006g for 5 min and re-
suspended in 200 ml of 95% methanol adjusted to pH = 2 using
concentrated HCl and incubated at 4uC for 18 hours before the
supernatant was collected by centrifugation at 180006g for
20 min. The methanol was evaporated using a rotary evaporator
and the crude extract was lyophilized and kept at 220uC for
further LC-MS/MS analysis.
11. Detection of Levan-sucrase by LC/MS-MSThe lyophilized extract was re-hydrated using 500 ml of 0.1%
formic acid and injected into Water Oasis HLB column
equilibrated with 0.1% formic acid. The sample was eluted with
600 ml 50% acetonitrile in 0.1% formic acid. The eluted sample
was separated by reversed phase chromatography using a Thermo
Scientific EASY-nLC II system with a reversed phase pre-column
Magic C18 AQ (100 mm I.D., 2 cm length, 5 mm, 100 A.
Michrom Bio Resources Inc, Auburn, CA) attached to nano-
analytical column Magic C18 (75 mm I.D., 15 cm length, 5 mm,
100 A. Michrom Bio Resources Inc, Auburn, CA). The flow rate
was set at 300 ml/min. The system was coupled to an LTQ
OrbitrapVelos mass spectrometer equipped with a Nanospray II
source (Thermo Fisher Scientific). Mobile phases were A (2%
acetonitrile in 0.1% formic acid) and B (90% acetonitrile in 0.1
formic acid). After a 249 bar (,5 ml) pre-column equilibration and
249 bar (,8 ml) nano-column equilibration, the sample was
separated by 55 min gradient as follows: (5% solvent B: 0 min,
40% solvent B: 60 min, 80% solvent B: 2 min and 80% solvent B:
8 min). The LTQ OrbitrapVelos (Thermo Fisher Scientific,
Bremen, Germany) parameters were as follows: Nano-electrospray
ion source with spray voltage 2.2 kV, capillary temperature
225uC, Survry MS1 scan m/z range from 400 to 2000 profile
mode, resolution 60,000 at 400 m/z with AGC target 1E6 and one
microscan with maximum inject time 200 ms. Lock mass Siloxane
445.120024 for internal calibration with preview mode for FTMS
master scan: on, injection waveforms: on, monoisotopic precursor
selection: on, rejection of charge state: 1. The sample was analysed
with a top-5 most intense ions charge state 2–4 exceeding 5000
counts were selected for CID FT-MSMS fragmentation and
detection in centroid mode. Dynamic exclusion setting were:
repeat count 2, repeat duration 15 seconds, exclusion list size 500,
exclusion duration 60 seconds with a 10 ppm mass window. The
CID activation isolation window was: 2 Da, AGC target: 1E4,
maximum inject time: 25 ms, activation time: 10 ms, activation Q:
0.250 and normalized collision energy 35%.
12. Data Analysis ParametersProteome Discoverer 1.3.0.339 software suite (Thermo Scien-
tific) was used to analyze raw files. Parameters for the spectrum
selection to generate peak lists of the CID spectra were as follows:
activation type: CID, s/n cut-off: 1.5, total intensity threshold: 0,
minimum peak count: 1, precursors mass: 350–5000 Da. The
peak lists were submitted to an in-hose mascot 2.2 against the
Uniprot-Swissprot databases (Figure 4).
Supporting Information
Figure S1 Comparison of regions for genes encoding levan-
sucrase (fructosyltransferase) enzyme in S. salivarius YU10 and S.
salivarius M18.
(TIF)
Text S1 Lantibiotic peptides detected in S. salivarius YU10
genome using SEED viewer software version 4.0.
(DOCX)
Text S2 DNA to protein translation of levan-sucrase or
fructosyltransferase (FTF) of S. salivarius YU10. Highlighted
residues are those detected by ESI-LC-MS/MS.
(DOCX)
Author Contributions
Conceived and designed the experiments: AB KP. Performed the
experiments: AB KP. Analyzed the data: AB KP. Contributed reagents/
materials/analysis tools: KP. Wrote the paper: AB KP.
References
1. Jack RW, Tagg JR, Ray B (1995) Bacteriocins of gram-positive bacteria.
Microbiol Rev 59: 171–200.
2. Cotter PD, Hill C, Ross RP (2005) Bacteriocins: developing innate immunity for
food. Nat Rev Microbiol 3: 777–788.
3. Abee T, Krockel L, Hill C (1995) Bacteriocins: modes of action and potentials in
food preservation and control of food poisoning. Int J Food Microbiol 28: 169–
185.
4. Lv W, Cong W, Cai Z (2004) Nisin production by Lactococcus lactis subsp. lactis
under nutritional limitation in fed-batch culture. Biotechnol Lett 26: 235–238.
5. Tramer J (1966) Nisin in food preservation. Chem Ind 11: 446–450.
6. Joo NE, Ritchie K, Kamarajan P, Miao D, Kapila YL (2012) Nisin, an
apoptogenic bacteriocin and food preservative, attenuates HNSCC tumorigen-
esis via CHAC1. Cancer Med 1: 295–305.
7. Karam L, Jama C, Nuns N, Mamede AS, Dhulster P, et al. (2013) Nisin
adsorption on hydrophilic and hydrophobic surfaces: evidence of its interactions
and antibacterial activity. J Pept Sci 19: 377–385.
8. Anderssen EL, Diep DB, Nes IF, Eijsink VG, Nissen-Meyer J (1998)
Antagonistic activity of Lactobacillus plantarum C11: two new two-peptide
bacteriocins, plantaricins EF and JK, and the induction factor plantaricin A.
Appl Environ Microbiol 64: 2269–2272.
9. Kristiansen PE, Fimland G, Mantzilas D, Nissen-Meyer J (2005) Structure and
mode of action of the membrane-permeabilizing antimicrobial peptide
pheromone plantaricin A. J Biol Chem 280: 22945–22950.
10. Holo H, Jeknic Z, Daeschel M, Stevanovic S, Nes IF (2001) Plantaricin W from
Lactobacillus plantarum belongs to a new family of two-peptide lantibiotics.
Microbiology 147: 643–651.
11. Chen S, Wilson-Stanford S, Cromwell W, Hillman JD, Guerrero A, et al. (2013)
Site-directed mutations in the lanthipeptide mutacin 1140. Appl Environ
Microbiol 79: 4015–4023.
12. Chattoraj P, Banerjee A, Biswas S, Biswas I (2010) ClpP of Streptococcus mutans
differentially regulates expression of genomic islands, mutacin production, and
antibiotic tolerance. J Bacteriol 192: 1312–1323.
13. Nguyen T, Zhang Z, Huang IH, Wu C, Merritt J, et al. (2009) Genes involved in
the repression of mutacin I production in Streptococcus mutans. Microbiology 155:
551–556.
14. Chaney N, Wilson-Stanford S, Kastrantas J, Dahal N, Smith L (2009) Rapid
method for extracting the antibiotic mutacin 1140 from complex fermentation
medium yeast extract. Can J Microbiol 55: 1261–1266.
15. Smith L, Hasper H, Breukink E, Novak J, Cerkasov J, et al. (2008) Elucidation of
the antimicrobial mechanism of mutacin 1140. Biochemistry 47: 3308–3314.
16. Wescombe PA, Upton M, Dierksen KP, Ragland NL, Sivabalan S, et al. (2006)
Production of the lantibiotic salivaricin A and its variants by oral streptococci
and use of a specific induction assay to detect their presence in human saliva.
Appl Environ Microbiol 72: 1459–1466.
17. Wescombe PA, Upton M, Renault P, Wirawan RE, Power D, et al. (2011)
Salivaricin 9, a new lantibiotic produced by Streptococcus salivarius. Microbiology
157: 1290–1299.
Lantibiotics Production by Streptococcus salivarius
PLOS ONE | www.plosone.org 15 June 2014 | Volume 9 | Issue 6 | e100541
18. Hyink O, Wescombe PA, Upton M, Ragland N, Burton JP, et al. (2007)
Salivaricin A2 and the novel lantibiotic salivaricin B are encoded at adjacent locion a 190-kilobase transmissible megaplasmid in the oral probiotic strain
Streptococcus salivarius K12. Appl Environ Microbiol 73: 1107–1113.
19. Birri DJ, Brede DA, Nes IF (2012) Salivaricin D, a novel intrinsically trypsin-resistant lantibiotic from Streptococcus salivarius 5M6c isolated from a healthy
infant. Appl Environ Microbiol 78: 402–410.20. Wescombe PA, Dyet KH, Dierksen KP, Power DA, Jack RW, et al. (2012)
Salivaricin G32, a homolog of the prototype Streptococcus pyogenes nisin-like
lantibiotic SA-FF22, produced by the commensal species Streptococcus salivarius.Int J Microbiol 2012: 738503.
21. Ross KF, Ronson CW, Tagg JR (1993) Isolation and characterization of thelantibiotic salivaricin A and its structural gene salA from Streptococcus salivarius
20P3. Appl Environ Microbiol 59: 2014–2021.22. Barretto C, Alvarez-Martin P, Foata F, Renault P, Berger B (2012) Genome
sequence of the lantibiotic bacteriocin producer Streptococcus salivarius strain K12.
J Bacteriol 194: 5959–5960.23. Heng NC, Haji-Ishak NS, Kalyan A, Wong AY, Lovric M, et al. (2011) Genome
sequence of the bacteriocin-producing oral probiotic Streptococcus salivarius strainM18. J Bacteriol 193: 6402–6403.
24. Islam MR, Nagao J, Zendo T, Sonomoto K (2012) Antimicrobial mechanism of
lantibiotics. Biochem Soc Trans 40: 1528–1533.25. Tabor AB (2011) The challenge of the lantibiotics: synthetic approaches to
thioether-bridged peptides. Org Biomol Chem 9: 7606–7628.26. van De Ven FJ, Jung G (1996) Structures of lantibiotics studied by NMR.
Antonie Van Leeuwenhoek 69: 99–107.27. Wilson-Stanford S, Smith L (2011) Commercial development and application of
type A lantibiotics. Recent Pat Antiinfect Drug Discov 6: 175–185.
28. Al-Mahrous MM, Upton M (2011) Discovery and development of lantibiotics;antimicrobial agents that have significant potential for medical application.
Expert Opin Drug Discov 6: 155–170.29. Smith L, Hillman J (2008) Therapeutic potential of type A (I) lantibiotics, a
group of cationic peptide antibiotics. Curr Opin Microbiol 11: 401–408.
30. Rathsam C, Giffard PM, Jacques NA (1993) The cell-bound fructosyltransferaseof Streptococcus salivarius: the carboxyl terminus specifies attachment in a
Streptococcus gordonii model system. J Bacteriol 175: 4520–4527.31. de Paula VC, Pinheiro IO, Lopes CE, Calazans GC (2008) Microwave-assisted
hydrolysis of Zymomonas mobilis levan envisaging oligofructan production.Bioresour Technol 99: 2466–2470.
32. Calazans GMT, Lopes CE, Lima RMOC, deFranca FP (1997) Antitumour
activities of levans produced by Zymomonas mobilis strains. Biotechnology Letters19: 19–21.
33. Leibovici J, Stark Y (1985) Increase in Cell-Permeability to a Cyto-Toxic Agent
by the Polysaccharide Levan. Cellular and Molecular Biology 31: 337–341.34. Dahech I, Belghith KS, Hamden K, Feki A, Belghith H, et al. (2011)
Antidiabetic activity of levan polysaccharide in alloxan-induced diabetic rats.
International Journal of Biological Macromolecules 49: 742–746.35. Belghith H, Song KB, Kim CH, Rhee SK (1996) Optimal conditions for levan
formation by an overexpressed recombinant levansucrase. Biotechnology Letters18: 467–472.
36. Jang KH, Song KB, Kim CH, Chung BH, Kang SA, et al. (2001) Comparison
of characteristics of levan produced by different preparations of levansucrasefrom Zymomonas mobilis. Biotechnology Letters 23: 339–344.
37. Han YW, Clarke MA (1990) Production and Characterization of MicrobialLevan. Journal of Agricultural and Food Chemistry 38: 393–396.
38. Barbour A, Philip K, Muniandy S (2013) Enhanced Production, Purification,Characterization and Mechanism of Action of Salivaricin 9 Lantibiotic
Produced by Streptococcus salivarius NU10. PLoS One 8: e77751.
39. Burton JP, Wescombe PA, Moore CJ, Chilcott CN, Tagg JR (2006) Safetyassessment of the oral cavity probiotic Streptococcus salivarius K12. Appl Environ
Microbiol 72: 3050–3053.40. Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, et al. (2008) The RAST
server: Rapid annotations using subsystems technology. BMC Genomics 9.
41. Tagg JR, Skjold SA (1984) A bacteriocin produced by certain M-type 49Streptococcus pyogenes strains when incubated anaerobically. J Hyg (Lond) 93: 339–
344.42. Wang Y (2010) Production and characterization of salivaricin MPS-like
inhibitory activity from Streptococcus uberis strain NY42 Thesis, Master of HealthSciences, University of Otago, Dunedin.
43. Somkuti GA, Gilbreth SE (2007) Influence of organic buffers on bacteriocin
production by Streptococcus thermophilus ST110. Curr Microbiol 55: 173–177.44. Tahara T, Kanatani K (1997) Isolation and partial characterization of crispacin
A, a cell-associated bacteriocin produced by Lactobacillus crispatus JCM 2009.FEMS Microbiol Lett 147: 287–290.
45. Mantovani HC, Hu HJ, Worobo RW, Russell JB (2002) Bovicin HC5, a
bacteriocin from Streptococcus bovis HC5. Microbiology-Sgm 148: 3347–3352.46. Kleerebezem M (2004) Quorum sensing control of lantibiotic production; nisin
and subtilin autoregulate their own biosynthesis. Peptides 25: 1405–1414.47. Tagg JR, Bannister LV (1979) ‘‘Fingerprinting’’ beta-haemolytic streptococci by
their production of and sensitivity to bacteriocine-like inhibitors. J MedMicrobiol 12: 397–411.
48. Wescombe PA, Burton JP, Cadieux PA, Klesse NA, Hyink O, et al. (2006)
Megaplasmids encode differing combinations of lantibiotics in Streptococcus
salivarius. Antonie Van Leeuwenhoek 90: 269–280.
Lantibiotics Production by Streptococcus salivarius
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